Many aquatic viruses such as Piscine orthoreovirus genotype 1 (PRV-1) are not culturable, necessitating genome assembly directly from samples. This approach is typically limited to samples with high viral load to ensure accuracy and adequate coverage during sequencing. Unfortunately, many natural infections and environmental substrates (e.g., seawater) have low viral loads. We adapted a recently developed multiplex PCR tiling method for targeted enrichment of Zika virus (Quick et al. 2017) to target the aquatic salmon virus PRV-1 from tissue and seawater and optimized library preparations for MinION sequencing. Specifically, PRV-1 from British Columbia was used as reference to design 36 sets of overlapping primer pairs using Primal Scheme software. RNA extraction and cDNA synthesis followed standard methods (Polinski et al. 2019) and was used for multiplex tiling PCR. Products were sequenced using MinKnow high-accuracy base calling and aligned using Nanopipe.
We sequenced 24-48 barcode-separated samples per flow cell, obtaining 2-3 million reads per sample. At this sequencing depth, near full-length genomes were obtained in samples with as low as 10 PRV-1 copies per µl with near-perfect agreement (>99.9%) to lllumina-derived assemblies for read depths of ≥ 20 reads per position. Samples which had received improper handling or were stored under suboptimal conditions often had significant loss of amplification across portions of the genome; however, even these typically yielded acceptable sequence coverage for most of the genome. By assembling over 300 PRV-1 genomes from a diverse set of fish and environmental samples, we were able to explore transmission pathways of PRV-1 including those within and between farmed and wild salmon over a large temporal range and spatial scale. We believe these methods are fundamentally suitable for application to a wide range of infectious agents in aquatic environments.